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Michael Jelínek METHODS FOR STUDYING OF PROTEINS I.

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Presentation on theme: "Michael Jelínek METHODS FOR STUDYING OF PROTEINS I."— Presentation transcript:

1 Michael Jelínek METHODS FOR STUDYING OF PROTEINS I

2 CONTENT OF THE LECTURE 1)Organization of the practice from Cell and Molecular Biology 2)General principles of specific detection of molecules 3)Microscopy techniques 4)Determination of protein concentration 5)Assessment of protein expression level SDS-PAGE + Western blot

3 1. ORGANIZATION OF THE PRACTISE 2 parts (2x 4 lectures) obligatory: for gaining credit is necessary 100% attendance and succesful passing of the practice test (writting during the second part of practice) absence (due to serious reasons) can be substituted after agreement of the head of practice by joining another studying group necessary equipment: lab coat, calculator necessary knowledge: theory from the lecture „Methods for studying of proteins I“ 1)Detection of actin and DNA in cancer cells by fluorescent microscopy 2) Comparison of protein expression in different tissues and samples by protein electrophoresis followed by Coomassie briliant blue stainning 2 practical tasks:

4 2.1. How to find specifically the target molecule among the thousands of other molecules? (necessary for the specifity of reaction) 2.2. How to visualize the result of the specific detection? Most of biological structures have no colour themself... (necessary to choose the proper system of signal detection) 2) GENERAL PRINCIPLES OF SPECIFIC DETECTION OF MOLECULES

5 2.1 SPECIFIC DETECTION OF TARGET MOLECULE Molecule in the sample is detected via specific and known interaction with another molecule The molecule interacting with target molecule can be: small organic molecule, which is known to bind specifically to our target molecule (e.g. taxol binds to tubulin, phalloidin binds to F-actin, DAPI and ethidium bromide bind to DNA) protein specifically recognizing target molecule, most widely antibody are used

6 2.1.1 Small organic molecule that can bind to target molecule Amanita Phaloidin Polymerisation of actin Polymerisation of tubulin Yew tree Paclitaxel DAPI, phaloidin, paclitaxel, nocodazol, ethidium bromid…

7 2.1.2 A. Proteins recognizing specifically cell structures Dnase I - binding of G actin in nucleus Concanavalin A - binding of glucose-manose groups, possible detection of cancer cells Annexin - binding of phosphatidylserine - detection of apoptotic cells

8 Antibodies recognizing any protein can be theoretically prepared Imunoglobulines produced by imunne system (Ig class A, D, E, G, M), recognize specific epitopes at the antigene molecule antigen = substance that induced production of antibodies directed to it by immune system, (usually a foreign molecule) epitope = specific area of the antigen surface that is bound by the antigen binding sites (part of antibody) Most widely used system for detection of proteins uses Antibodies = IMUNODETECTION B. Proteins recognizing specifically cell structures - antibodies Light chain Haevy chain Antigen binding sites

9 2.2. POSSIBILITIES OF SIGNAL DETECTION Detecting molecule (specifically recognizing target molecule) is usually not possible to be directly observed (exception e.g. DAPI) Thus, detecting molecule must be conjugated with another molecule that is able to produce detectable signal Such conjugates can be: Heavy metal Fluorophore Enzyme whose enzymatic activity enable visualization of detected molecule - light, colour signal

10 Labeled primary antibody (direct detection) vs Non-labeled primary and labeled secondary antibody (indirect detection) Direct and indirect (immuno)detection Signal amplification substrate enzyme detectable product Direct detectionIndirect detection detectable product substrate enzyme primary antibody secondary antibody

11 2.2.1 Vizualisation by conjugated heavy metal Production of somatostatin in pancreatic cells

12 fluorophore = molecule that is able to absorb light of a specific wavelength and emits light at a longer wavelength (=signal we detect) Vizualisation by conjugated fluorophore

13 Visualization of target molecule after reaction of enzyme with its substrate chemiluminiscence - enzyme horse radish peroxidase (= HRP) is usually used. HRP cleaves hydroxide peroxide and created radicals activate luminol - light is emited and detected (by special instrument or photography film e. g. western blot) chromogenic (color reaction) - enzyme produces coloured product (colorimetr, e.g. ELISA) Visualization by the activity of conjugated enzyme product must be insoluble if the localization of target molecule is detected - imunofluorescence product must be soluble if intensity of the colour in solution is measured (quantification of the target molecule via measurement of absorbance)

14 3) MICROSCOPY TECHNIQUES 3.1 Imunohistochemistry 3.2 Electron mikroscopy 3.3 Fluorescence microscopy

15 3.1. IMUNOHISTOCHEMISTRY detection in microscopy preparat (thin tissue section) often used usually specific antibodies used and chromogenic reaction or fluorescence show localisation of the target protein Insulin in cells of pancreas

16 3.2. ELECTRON MICROSCOPY Somatostatin in D cells in pancreas Not so often used

17 3.3. FLUORESCENCE MICROSCOPY Using of single fluorophore vs using of more fluorophores Using of various fluorophores - possibility of observation of more types of molecules as well as their colocalisation (presence on the same cells or at the same place in the cell) At the practise: actin and DNA stainning The signal only from one fluorophore can be observed in a microscope, for multicolour stainning multicolour picture must be assembled from single colored pictures by specific software  DNA: stained by DAPI  tubulin: stained by anti-tubülin antibody conjugated with fluorophore FITC  actin: stained by phalloidin conjugated with fluorophore TRITC

18 4) DETERMINATION OF THE CONCENTRATION OF PROTEINS IN SOLUTION based on spectrophotometry (measurement of absorbance) 4.1 Determination from absorbance in UV spectrum 4.2 BCA assay (Bicinchonic assay) 4.3 Bradford assay (will be used in practice)

19 4.1. Determination from absorbation in UV spectrum Proteins naturally exhibit absorption in UV part ( nm) of spectrum - primarily due to presence of aromatic aminoacids (tyrosine and tryptophan) no need for calibration curve Calculation: [Protein] (mg/mL) = 1.55*A *A260 !!! all methods that are based on detection of presence of only certain types of aminoacids in samples are reliable only if there is no big difference in aminoacid composition in proteins of analyzed samples !!!

20 Detection reagent contains BCA (bicinchoninic acid) Colorimetric reaction results from interaction of BCA with peptide bond (and not only from interaction with specific aminoacids) Intensity of purple colour is measured at 562 nm 4.2. BCA assay Protein concentration

21 4.3. BREDFORD ASSAY Principle: colorimetric reaction after mixing of Bradford reagent with proteins containing solution Bradford reagent contains Coomassie Brilliant Blue - binds to basic and aromatic AA in proteins (Arg, Phe, Try, Pro) Presence of proteins changes the colour of solution from brown to blue Absorbance is measured at 595 nm (absorbance correlated with concentration of proteins) Protein concentration

22 QUANTIFICATION OF PROTEINS IN SAMPLES construction of calibration curve using samples with KNOWN concentration of protein - usually serial dilution of bovine serum albumin (BSA) Measured value of absorbance Corresponding protein concentration

23 5) ASSESSMENT OF PROTEIN EXPRESSION LEVEL Metods based only on immunodetection: ELISA, flow cytometry SDS-PAGE + Western blot SDS-PAGE - Method used for separation of proteins according to their molecular weight = sodium dodecyl sulphate polyacrylamide gel electrophoresis used for determination (comparison) of protein expression in samples sample in the form of protein-containing solution, e.g. tissue lysate, … before analysis, desintegration of cells (= cell lysis) is necessary to release the cell content into solution cell lysis is performed usually by various chemical, mechanical and physical approaches, or by their combination

24

25 5.1. Preparation of samples for separation, proteins must be denaturated into individual polypeptide chains Proteins must be denaturated by denaturatig agens before separation SDS (sodium dodecyl sulfate) wih heating are used releasing proteins into fibre form merkaptoethanol, dithiotreitol - reduce S-S bridges They also have homogenic negative charge due to SDS treatment, the same for length unit of protein

26 gel electrophoresis  gel with such pore size that enables molecules with different size to move with different speed proteins: usually polyacrylamide gel (electrophoresis of DNA, RNA: usually agarose gel - bigger pore size than acrylamide gel) 5.2 Principle of electrophoresis

27 Separation of proteins only according to molecular weights, proteins are negatively charged - they move in electric field to anode - positive electrode negatively charged proteins move to positive electrode, longer polypeptides chains are more retarded by the gel structure than shorter chains  shorter chains travel faster molecular weight of protein of our interest can be found by comparing its size with molecular weight marker = commercially available mixture of proteins with known molecular masses 5.3 Course of electrophoresis

28 5.4 Gel stainning Blue stain Coomassie blue binds nespecifically all proteins (see Bredford method), each strip (band) represents a group of proteins with certain size Marker Increasing concentration of protein

29 5.5. Western blot SOUTHERN blot (technique for DNA detection) developed by Edwine Southern (1975) NORTHERN blot (technique for RNA detection) WESTERN blot: Used usually for comparison of protein expression using immunodetection Transfer of proteins separated by SDS- PAGE to a protein-binding surface (nitrocelulose or PVDF membrane) negatively charged proteins move from polyacrylamide gel to the membrane membrane enables easy manipulation and easy access of antibodies during next steps of the target protein detection

30 proteins stick to the membrane in the same position they had in the gel membrane blocking (usually BSA or defatted milk) - saturation of all protein binding-sites on the membrane detection of target protein by specific antibodies detection of protein of our interest usually by specific antibodies and chemiluminiscent or chromogenic reaction with formation of insoluble product

31 Membrane strips containing electrophoretically separated antigen extracts are used as solid phase. The position of the proteins depends on their respective molecular masses. If the pacient´s sample is positive, specific antibodies in his serum attach to the antigens coupled to the membrane. The attached antibodies react with AP-labelled anti-human antibodies (AP = alkaline phosphatase). The enzyme activity than mark the sites where is the antibody bound (if it really is), 5.6. Medicinal application presence of the antigen in blood of pacient is finally proved/disproved

32 Detection of antibodies (e.g. for confirmation of diagnosis) boreliosis, EBV, HIV, HSV, Helicobacter pylori autoantibodies, antibodies against nuclear antigens (ANA), antibodies against neural antigens


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